This invention relates to blood staining systems and, more particularly, to the stabilization of Romanowsky-type stains, e.g., Wright's solution and Giemsa's solution. These stains comprise a methanol solution of Methylene Blue and an eosin dye along with other allied dyes. Typical allied dyes include Azure A, Azure B and Azure C with Eosin Y being the preferred eosin dye which is typically introduced to the methanol solution in the form of its disodium salt.
In order to employ such a stain, it is usually necessary to prepare a solution of the dry stain in methyl alcohol and apply the solution to a blood smear or the like. Next, a buffer solution and a rinse solution are added stepwise to the stained smear until a differential stain of cells occurs on the smear when observed with the microscope.
The presence of a second dye, in addition to Methylene Blue, particularly Eosin Y, is desirable to enhance the staining qualities of the solution. Other allied dyes, i.e., Azure A, Azure B and Azure C are also desirable for their enhancement of the solution's ability to stain the blood smear. The standard way of using such dyes is to form the methanol solution and allow it to stand for a period of time. The azure dyes in the stain powder are not very soluble in methanol, and Methylene Blue degrades into azures by a demethylation reaction in the presence of eosin upon aging in solution. It will normally take about two weeks of standing for the optimal staining result to be achieved. Unfortunately, the dyes continue to degrade, and the resultant degradation products render the solution unsuitable for the intended purpose. Furthermore, random precipitation in the stain solution upon aging results in poor stain quality. Thus, while the stain solution takes about two weeks to become fully effective, it has a shelf life of only about 3 to approximately 12 months.
Gilliland et al report in Stain Technology, Vol. 54, No. 3, pp. 141-150 that a Romanowsky-type blood stain can be stabilized by making the solution acidic to protonate the eosin thereby inhibiting the formation of the precipitate. For use, the stain is neutralized by a specially formulated fixative solution. We have found that this technique is not suitable for the stabilization of dyes of the concentration necessary for suitability in certain automated staining devices. This concentration should be at least sufficient to provide a solution which, when scanned by spectrophotometer, will absorb 230 optical density units at 645 nanometers (nm) and 128 optical density units at 525 nm because of the required short staining time in such automated devices. As used herein, an optical density unit is defined as the absorbance multiple of the dilution factor, i.e., it is a function of concentration. For example, a solution which exhibited an absorbance by spectrophotometric measurement of 0.575 at a wavelength of 645 nm after it had been diluted 400 times would absorb 0.575.times.400 or 230 optical density units. Absorption measurements are made at these wavelengths because a solution of Methylene Blue and azures gives an absorption maximum at 645 nm and, a solution of Eosin Y gives an absorption maximum at 525 nm. While Gilliland et al do not disclose the concentration of their stain solution in the cited article, judging from the absorbance data they disclose in their FIG. 1, the concentration of their stain is estimated to be at a level of optical density of about 162 optical density units at 645 nm and 87.5 optical density units at 525 nm which is about 70% of the concentration required for suitability of the solution in automated staining devices referred to above. In acidifying stain solutions of the required concentration by the addition of HCl, it has been found that acidification to pH 3.0 as disclosed by Gilliland et al is insufficient to prevent precipitation in the more concentrated staining solutions which are useful in the automatic staining devices. Further acidification to pH 1.6 will prevent precipitation but at this pH, the Eosin Y is irreversibly converted to some other species which degrades the staining performance of the dye.
Marshall et al disclose in J. Clin. Pathol., 28: 920923 (1975) the preparation of a Romanowsky-type stain by dissolving Methylene Blue, Azure B and the free acid of an eosin dye in a 1:1 v/v mixture of glycerol and methanol. They describe this solution as being extremely stable although they report that conventional Romanowsky-type stains show similar stability. In any event, a stain solution in which the solvent is 50% glycerol is known to be highly stable but has such a high viscosity that it is unsuitable for use in automated staining devices.